Lack of Charge Interaction in the Ion Binding Site Determines Anion Selectivity in the Sodium Bicarbonate Cotransporter NBCe1

The Na/HCO3 cotransporter NBCe1 is a member of SLC4A transporters that move HCO3− across cell membranes and regulate intracellular pH or transepithelial HCO3 transport. NBCe1 is highly selective to HCO3− and does not transport other anions; the molecular mechanism of anion selectivity is presently unclear. We previously reported that replacing Asp555 with a Glu (D555E) in NBCe1 induces increased selectivity to other anions, including Cl−. This finding is unexpected because all SLC4A transporters contain either Asp or Glu at the corresponding position and maintain a high selectivity to HCO3−. In this study, we tested whether the Cl− transport in D555E is mediated by an interaction between residues in the ion binding site. Human NBCe1 and mutant transporters were expressed in Xenopus oocytes, and their ability to transport Cl− was assessed by two-electrode voltage clamp. The results show that the Cl− transport is induced by a charge interaction between Glu555 and Lys558. The bond length between the two residues is within the distance for a salt bridge, and the ionic strength experiments confirm an interaction. This finding indicates that the HCO3− selectivity in NBCe1 is established by avoiding a specific charge interaction in the ion binding site, rather than maintaining such an interaction.


Introduction
NBCe1 is a membrane protein that mediates electrogenic Na + -HCO 3 − and/or CO 3 2− transport across cell membrane and regulates intracellular and extracellular pH, as well as transepithelial HCO 3 − transport in many cells [1][2][3][4]. NBCe1 was first physiologically identified in the kidney proximal tubules [5], where it is responsible for reabsorbing two thirds of filtered HCO 3 − . NBCe1 is highly selective to HCO 3 − and does not transport other anions, including Cl − [5]. The gene encoding NBCe1 was expression-cloned in the late 1990s by Romero et al. [1]; since then, it has provided valuable information on the molecular and cellular physiology of Na/HCO 3 transport mediated by NBCe1 and its family proteins, collectively the Na + -coupled bicarbonate transporters, in humans. NBCe1 exists as multiple variants, due to different N-and C-terminal sequences, and each variant differs in tissue expression, intrinsic functional properties, and regulation [6]. The transporter has a Na + :HCO 3 − stoichiometry of 1:3 in renal proximal tubules and 1:2 in other cells, as well as in heterologous expression systems. Overall, NBCe1 plays an important role in the physiology and pathophysiology of many different organs, such as kidneys, heart, brain, eyes, enamel organs, and intestines [7][8][9][10].
The cryoEM structure of NBCe1 [11] has provided details on the protein structure and amino acid residues responsible for ion transport. NBCe1 is a homodimer with each monomer, consisting of 14 transmembrane segments (TMs), extracellular loops, and cytoplasmic regions. TMs 5-7 and 12-14 form the gate domain and TMs 1-4 and 8-11 form the core domain, while the cavity between these two domains houses an ion access pathway, through which Na + and CO 3 2− (HCO 3 − ) move. The ion accessibility pathway

I Cl Produced by D555E
To record I Cl , produced by D555E, we expressed human NBCe1 and mutant D555E in oocytes and applied them with 71 mM Cl − during superfusion of Cl − -free solution. Figure 1A shows a representative current trace, produced by NBCe1, in voltage clamp (the holding potential of −60 mV). NBCe1 did not produce measurable I Cl , in response to bath Cl − , but produced an outward I NBC upon exposure to 5% CO 2 , 25 mM HCO 3 − , consistent with its electrogenic Na/HCO 3 cotransport activity. In contrast, D555E produced an outward I Cl , in response to Cl − ( Figure 1B). I Cl was markedly decreased in the presence of CO 2 /HCO 3 − , consistent with our previous report [17] that D555E produces I Cl , which can be inhibited by HCO 3 − . Mean I Cl from other oocytes (n = 6/group) is summarized in Figure 1C. On average, 70% of I Cl produced by D555E was reduced in the presence of CO 2 /HCO 3 − (p < 0.01, two-way ANOVA). In other experiments, we then determined I-V relationships for I Cl and I NBC to compare the current responses at different voltages in NBCe1 vs. D555E. As shown in Figure 1D, D555E evoked large outward currents at positive potentials in ND96 solution containing 96 mM Cl − (p < 0.01, n = 5), reflecting Cl − influx.
CO2/HCO3 − (p < 0.01, two-way ANOVA). In other experiments, we then determined I-V relationships for ICl and INBC to compare the current responses at different voltages in NBCe1 vs. D555E. As shown in Figure 1D, D555E evoked large outward currents at positive potentials in ND96 solution containing 96 mM Cl − (p < 0.01, n = 5), reflecting Cl − influx. However, in CO2/HCO3 − solution ( Figure 1E), NBCe1 produced larger outward INBC than D555E at positive potentials (p < 0.05, n = 5). The two I-V curves were parallel to each other in the outward direction, as they are INBC. The curves crossed at a negative potential (approximately −80 mV), probably due to Cl − efflux via D555E in the inward direction. NBCe1. An oocyte expressing NBCe1 was superfused with modified Cl − -free ND96 until the basal current became stable, and then exposed to 71 mM Cl − before and after switching solutions equilibrated with 5% CO2, 25 mM HCO3 − . The holding potential was −60 mV. (B) Representative ICl and INBC, produced by D555E. The recording was performed, as described in (A). (C) Mean ICl, in the absence and presence of CO2/HCO3 − . ICl was measured when the current reached steady-state after Cl − application (n = 6/group); ** p < 0.01 compared to ICl in the absence of CO2/HCO3 − . (D,E) I-V relationships of NBCe1 and D555E for ICl in ND96 (D) and CO2/HCO3 − (E). Currents were obtained by a step voltage command from −120 to +60 mV with 20 mV increments (n = 5/group).

Na + Prerequisite for HCO3 − to Access Its Binding Site
The above results reveal that Cl − transport by D555E is less favorable than HCO3 − transport, when both ions are present in the bath. To determine whether this feature depends on Na + , we performed two sets of experiments. In the first set of experiments, we recorded ICl in Na + -free CO2/HCO3 − solution and tested whether ICl could be reduced under this condition. Representative recordings of ICl, produced by NBCe1 and D555E, are shown in Figure 2A,B. In contrast to NBCe1, D555E produced ICl in the absence of CO2/HCO3 − and, more importantly, in the Na + -free CO2/HCO3 − solution. The current amplitudes were similar in both solutions, indicating that HCO3 − has negligible effect on ICl under the Na + -free condition. Figure 2C is a comparison of the mean ICl between groups in these two solutions from other oocytes (n = 5 NBCe1 and 10 D555E). No significant difference was observed within groups. In the second set of experiments, we induced ICl in Na + -free solution and tested whether the induced ICl could remain after CO2/HCO3 − application under the continued Na + -free condition. As shown in Figure 2D,E, whereas NBCe1 had no ICl, D555E produced ICl under the Na + -free condition, regardless of bath Representative I Cl and I NBC produced by NBCe1. An oocyte expressing NBCe1 was superfused with modified Cl − -free ND96 until the basal current became stable, and then exposed to 71 mM Cl − before and after switching solutions equilibrated with 5% CO 2 , 25 mM HCO 3 − . The holding potential was −60 mV. (B) Representative I Cl and I NBC , produced by D555E. The recording was performed, as described in (A). (C) Mean I Cl , in the absence and presence of CO 2 /HCO 3 − . I Cl was measured when the current reached steady-state after Cl − application (n = 6/group); ** p < 0.01 compared to I Cl in the absence of CO 2 /HCO 3 − . (D,E) I-V relationships of NBCe1 and D555E for I Cl in ND96 (D) and CO 2 /HCO 3 − (E). Currents were obtained by a step voltage command from −120 to +60 mV with 20 mV increments (n = 5/group).

Na + Prerequisite for HCO 3 − to Access Its Binding Site
The above results reveal that Cl − transport by D555E is less favorable than HCO 3 − transport, when both ions are present in the bath. To determine whether this feature depends on Na + , we performed two sets of experiments. In the first set of experiments, we recorded I Cl in Na + -free CO 2 /HCO 3 − solution and tested whether I Cl could be reduced under this condition. Representative recordings of I Cl , produced by NBCe1 and D555E, are shown in Figure 2A,B. In contrast to NBCe1, D555E produced I Cl in the absence of CO 2 /HCO 3 − and, more importantly, in the Na + -free CO 2 /HCO 3 − solution. The current amplitudes were similar in both solutions, indicating that HCO 3 − has negligible effect on I Cl under the Na + -free condition. Figure 2C is a comparison of the mean I Cl between groups in these two solutions from other oocytes (n = 5 NBCe1 and 10 D555E). No significant difference was observed within groups. In the second set of experiments, we induced I Cl in Na + -free solution and tested whether the induced I Cl could remain after CO 2 /HCO 3 − application under the continued Na + -free condition. As shown in Figure 2D,E, whereas NBCe1 had no I Cl , D555E produced I Cl under the Na + -free condition, regardless of bath CO 2 /HCO 3 − . A slight decrease after CO 2 /HCO 3 − application is probably due to Cl − mismatch between solutions. Consistent with this result, comparison of mean I Cl (n = 5/group), before and after CO 2 /HCO 3 − application, resulted in no significant difference ( Figure 2F). Conclusively, the results from the two sets of experiments demonstrate D555E preference to Cl − over HCO 3 − in the absence of Na + , implying that Na + is required for HCO 3 − to access its binding site.
CO2/HCO3 − . A slight decrease after CO2/HCO3 − application is probably due to Cl − mismatch between solutions. Consistent with this result, comparison of mean ICl (n = 5/group), before and after CO2/HCO3 − application, resulted in no significant difference ( Figure 2F). Conclusively, the results from the two sets of experiments demonstrate D555E preference to Cl − over HCO3 − in the absence of Na + , implying that Na + is required for HCO3 − to access its binding site. ICl was measured before and after CO2/HCO3 − was applied. All solutions lacked Na + . (F) Mean ICl before and after application of CO2/HCO3 − under the Na + -free condition (n = 5/group). The holding potential was −60 mV in all experiments.

Lack of ICl in the TM5-Replaced Chimeric Transporter
D555E is charge-conserved but has different geometry of the carboxyl group in the side chain due to an additional carbon backbone. This led us to postulate that Glu 555 in D555E interacts with a nearby residue which results in a gain of function to select Cl − . To investigate this possibility, we replaced NBCe1/TM5 with NBCn1/TM5, which contains a Glu at the corresponding site of Asp 555 , and measured ICl in the chimeric transporter. First, we determined the functionality of the chimeric transporter by simultaneous recording of pHi and INBC in voltage clamp ( Figure 3A,B). In oocytes expressing NBCe1, the pHi initially decreased upon CO2/HCO3 − application, due to CO2 influx followed by H + accumulation from hydration ( Figure 3A). The pHi was then recovered from an acidification (arrow) as HCO3 − is continuously transported into the oocyte by NBCe1 and associates with intracellular H + . Applying CO2/HCO3 − also elicited an outward INBC (arrowhead), consistent with an influx of a net negative charge, due to 1 Na + and 2 HCO3 − (or 1 CO3 2− ). Figure 3B is a recording of pHi and INBC, produced by the TM5-replaced chimeric transporter, subjected to the same experimental protocol. The chimeric transporter had a slower pHi recovery rate (dpH/dt) and smaller INBC in CO2/HCO3 − solution than NBCe1. Consistent with this observation, mean dpH/dt and INBC from 5 oocytes per group were significantly decreased in the chimeric transporter (p < 0.01 for each; Figure 3C,D). Despite such decreases, the chimeric transporter is functional, as it recovers pHi from an acidification and D555E. I Cl was measured before and after CO 2 /HCO 3 − was applied. All solutions lacked Na + .
(F) Mean I Cl before and after application of CO 2 /HCO 3 − under the Na + -free condition (n = 5/group).
The holding potential was −60 mV in all experiments.

Lack of I Cl in the TM5-Replaced Chimeric Transporter
D555E is charge-conserved but has different geometry of the carboxyl group in the side chain due to an additional carbon backbone. This led us to postulate that Glu 555 in D555E interacts with a nearby residue which results in a gain of function to select Cl − . To investigate this possibility, we replaced NBCe1/TM5 with NBCn1/TM5, which contains a Glu at the corresponding site of Asp 555 , and measured I Cl in the chimeric transporter. First, we determined the functionality of the chimeric transporter by simultaneous recording of pH i and I NBC in voltage clamp ( Figure 3A,B). In oocytes expressing NBCe1, the pH i initially decreased upon CO 2 /HCO 3 − application, due to CO 2 influx followed by H + accumulation from hydration ( Figure 3A). The pH i was then recovered from an acidification (arrow) as HCO 3 − is continuously transported into the oocyte by NBCe1 and associates with intracellular H + . Applying CO 2 /HCO 3 − also elicited an outward I NBC (arrowhead), consistent with an influx of a net negative charge, due to 1 Na + and 2 HCO 3 − (or 1 CO 3 2− ). Figure 3B is a recording of pH i and I NBC , produced by the TM5-replaced chimeric transporter, subjected to the same experimental protocol. The chimeric transporter had a slower pH i recovery rate (dpH/dt) and smaller I NBC in CO 2 /HCO 3 − solution than NBCe1. Consistent with this observation, mean dpH/dt and I NBC from 5 oocytes per group were significantly decreased in the chimeric transporter (p < 0.01 for each; Figure 3C,D). Despite such decreases, the chimeric transporter is functional, as it recovers pH i from an acidification and produces I NBC . Next, we measured the I Cl and I NBC produced by the chimeric transporter. Interestingly, the chimeric transporter did not produce measurable I Cl , while retaining I NBC ( Figure 3E; one of 9 oocytes expressing the chimeric transporter is shown). Consistent with this result, I-V relationships exhibited negligible change in curves before and after Cl − application (p > 0.05, n = 5; Figure 3F). Figure 3G is the comparison of Cl − conductance (G Cl ), calculated from the slope of the I Cl -V relationship (i.e., difference in I-V curve before and after Cl − application). G Cl of the chimeric transporter was negligible. and produces INBC. Next, we measured the ICl and INBC produced by the chimeric transporter. Interestingly, the chimeric transporter did not produce measurable ICl, while retaining INBC ( Figure 3E; one of 9 oocytes expressing the chimeric transporter is shown). Consistent with this result, I-V relationships exhibited negligible change in curves before and after Cl − application (p > 0.05, n = 5; Figure 3F). Figure 3G is the comparison of Cl − conductance (GCl), calculated from the slope of the ICl-V relationship (i.e., difference in I-V curve before and after Cl − application). GCl of the chimeric transporter was negligible.

ICl Induced by Lys 558 Replacement in the TM5 Chimeric Transporter
The result of negligible ICl in the chimeric transporter indicates that Glu 555 is not the sole residue for ICl and additional residues are involved. Those residues should be in TM5 because other TMs were unchanged in the chimeric transporter. Asp 555 is a residue in the anion binding site S2 that includes Lys 558 , Lys 559 , and Lys 562 ( Figure 4A). The chimeric transporter contains Glu 555 , Glu 558 , Lys 559 , and Asp 562 at the corresponding sites ( Figure 4B), suggesting that residues at position 558 and 562 would be responsible for ICl. To test this possibility, we changed Glu 558 and Asp 562 , individually or together, in the chimeric transporter with a Lys and tested their ability to produce ICl (n = 4-5/group). Figure 4C shows the I-V relationships for the chimeric transporter without mutation. As expected, no significant difference was observed in the I-V curves before and after Cl − application (red line in the figure). In contrast, replacing Glu 558 with a Lys (E558K) increased the slope . Data were averaged from 5 oocytes per group. (G) Mean Cl − conductance, G Cl . G Cl was calculated from slopes in I Cl -V curve, which is the difference in I-V relationships between the presence and absence of Cl − in (F). Slopes were measured near zero-current potentials; ** p < 0.01.

I Cl Induced by Lys 558 Replacement in the TM5 Chimeric Transporter
The result of negligible I Cl in the chimeric transporter indicates that Glu 555 is not the sole residue for I Cl and additional residues are involved. Those residues should be in TM5 because other TMs were unchanged in the chimeric transporter. Asp 555 is a residue in the anion binding site S2 that includes Lys 558 , Lys 559 , and Lys 562 ( Figure 4A). The chimeric transporter contains Glu 555 , Glu 558 , Lys 559 , and Asp 562 at the corresponding sites ( Figure 4B), suggesting that residues at position 558 and 562 would be responsible for I Cl . To test this possibility, we changed Glu 558 and Asp 562 , individually or together, in the chimeric transporter with a Lys and tested their ability to produce I Cl (n = 4-5/group). Figure 4C shows the I-V relationships for the chimeric transporter without mutation. As expected, no significant difference was observed in the I-V curves before and after Cl − application (red line in the figure). In contrast, replacing Glu 558 with a Lys (E558K) increased the slope in the outward direction upon Cl − application ( Figure 4D). Replacing Asp 562 with a Lys (D562K) had no effect ( Figure 4E) and displayed similar I-V curves as the chimeric transporter. Consistent with these results, replacing both Glu 558 and Asp 562 with Lys (E558K/D562K) increased the slope in the outward direction upon Cl − application ( Figure 4F). Thus, Lys 558 is responsible for producing I Cl .
in the outward direction upon Cl − application ( Figure 4D). Replacing Asp 562 with a Lys (D562K) had no effect ( Figure 4E) and displayed similar I-V curves as the chimeric transporter. Consistent with these results, replacing both Glu 558 and Asp 562 with Lys (E558K/D562K) increased the slope in the outward direction upon Cl − application ( Figure  4F). Thus, Lys 558 is responsible for producing ICl. Next, we compared ICl and INBC produced by the mutant transporters. The chimeric transporter without mutation had negligible ICl but produced measurable INBC ( Figure 5A). A transitional undershoot after Cl − washout is probably due to endogenous Cl − efflux which often occurs in some preparations of oocytes. E558K and E558K/D562K produced ICl ( Figure 5B,D), whereas D562K did not ( Figure 5C). Both E558K and E558K/D562K showed higher ICl amplitudes than INBC amplitudes, the reason of which is unclear. Comparison of mean ICl from 5-6 oocytes per group is summarized in Figure 5E. A significant amount of ICl was produced when a Lys was present at position 558 (p < 0.01, one-way ANOVA). We also compared mean INBC between groups to evaluate the effect of Lys mutations on Na/HCO3 cotransport and found a decrease in INBC by the mutations (p < 0.01, one-way ANOVA; Figure 5F). Thus, positively charged Lys residues in site S2 appear to have negative effects on the transporter activity. Next, we compared I Cl and I NBC produced by the mutant transporters. The chimeric transporter without mutation had negligible I Cl but produced measurable I NBC ( Figure 5A). A transitional undershoot after Cl − washout is probably due to endogenous Cl − efflux which often occurs in some preparations of oocytes. E558K and E558K/D562K produced I Cl ( Figure 5B,D), whereas D562K did not ( Figure 5C). Both E558K and E558K/D562K showed higher I Cl amplitudes than I NBC amplitudes, the reason of which is unclear. Comparison of mean I Cl from 5-6 oocytes per group is summarized in Figure 5E. A significant amount of I Cl was produced when a Lys was present at position 558 (p < 0.01, one-way ANOVA). We also compared mean I NBC between groups to evaluate the effect of Lys mutations on Na/HCO 3 cotransport and found a decrease in I NBC by the mutations (p < 0.01, oneway ANOVA; Figure 5F). Thus, positively charged Lys residues in site S2 appear to have negative effects on the transporter activity.

Salt Bridge between Glu 555 and Lys 558
The identification of Lys 558 for I Cl leads to the possibility of a charge interaction between Glu 555 and Lys 558 . To test whether a salt bridge stability is involved, we compared I Cl produced by E558K in solutions containing either low or high ionic strength. The solution osmolarity was maintained using mannitol. The chimeric transporter displayed negligible response to 1-96 mM Cl − in superfusing solutions, with the ionic strength of 0.005 and 0.1 mol/L ( Figure 6A,C), consistent with its lack of I Cl . In contrast, E558K produced I Cl with progressively larger amplitudes at higher NaCl concentrations when measured in solutions with the ionic strength of 0.005 mol/L ( Figure 6B) but had nearly negligible I Cl , when measured in solutions with the ionic strength of 0.1 mol/L ( Figure 6D). The result is consistent with the fact that a favorable salt bridge is diminished by a high ionic strength [21]. The decreasing effect by a high ionic strength was evident from the graph of I Cl plotted as a function of Cl − concentration ( Figure 6E). The result shows effective inhibition of E558K-mediated I Cl by a high ionic strength (F 12,80 = 7.47, p < 0.01 for transporter x Cl − concentration interaction, two-way ANOVA; n = 4-6/group).  Figure 1. (E) Mean ICl produced by the mutants. The level of significance was determined using one-way ANOVA, with Sidak post-test (n = 5-6/group). (F) Mean INBC produced by the mutants. Peak currents after CO2/HCO3 − application were measured. * p < 0.05 and ** p < 0.01 compared to TM5.

Salt Bridge between Glu 555 and Lys 558
The identification of Lys 558 for ICl leads to the possibility of a charge interaction between Glu 555 and Lys 558 . To test whether a salt bridge stability is involved, we compared ICl produced by E558K in solutions containing either low or high ionic strength. The solution osmolarity was maintained using mannitol. The chimeric transporter displayed negligible response to 1-96 mM Cl − in superfusing solutions, with the ionic strength of 0.005 and 0.1 mol/L ( Figure 6A,C), consistent with its lack of ICl. In contrast, E558K produced ICl with progressively larger amplitudes at higher NaCl concentrations when measured in solutions with the ionic strength of 0.005 mol/L ( Figure 6B) but had nearly negligible ICl, when measured in solutions with the ionic strength of 0.1 mol/L ( Figure 6D). The result is consistent with the fact that a favorable salt bridge is diminished by a high ionic strength [21]. The decreasing effect by a high ionic strength was evident from the graph of ICl plotted as a function of Cl − concentration ( Figure 6E). The result shows effective inhibition of E558K-mediated ICl by a high ionic strength (F12,80 = 7.47, p < 0.01 for transporter x Cl − concentration interaction, two-way ANOVA; n = 4-6/group).

Glu 555 -Lys 558 Charge Interaction
To further determine the above salt bridge interaction, we analyzed the bond length between the carboxyl group in the side chain of Glu 555 and the amino group of Lys 558 using the structure editing function with Dunbrack rotamer library [22] built in ChimeraX. In

Glu 555 -Lys 558 Charge Interaction
To further determine the above salt bridge interaction, we analyzed the bond length between the carboxyl group in the side chain of Glu 555 and the amino group of Lys 558 using the structure editing function with Dunbrack rotamer library [22] built in ChimeraX. In NBCe1, the distance between the carboxyl group of Asp 555 and the amino group of Lys558 was 5.63 Å (Figure 7A), higher than the maximum 4.0 Å required for a hydrogen bond [23]. However, in D555E, the bond length between Glu 555 and Lys 558 was 3.79 Å ( Figure 7B). The lengths from Lys 559 and Lys 562 were higher than 4.0 Å (data not shown). Thus, the bond length was consistent with a weak electrostatic interaction between Glu 555 and Lys 558 , but neither Lys 559 nor Lys 562 . The importance of the Glu 555 /Lys 558 interaction for I Cl was further examined by replacing Glu 555 and Lys 558 residues with other amino acids and comparing their ability to produce I Cl ( Figure 7C). Replacing Glu 555 with a neutral Asn or Gln (N-K and Q-K pairs in the figure) or Lys 558 with an Asp (E-E) near completely abolished I Cl . In contrast, replacing Lys 558 with a positively charged Arg (E-R) retained measurable I Cl . One-way ANOVA, with Sidak post-test, revealed a significant change in I Cl for N-K, Q-K and E-E pairs compared to E-K and E-R pairs (F 5,24 = 25.49, p < 0.01; n = 4-7/group). Water-injected control showed no current. . N-K and Q-K are the replacement of Glu 555 with an asparagine and a glutamine, respectively (n = 5/group). E-E and E-R are the replacement of Lys 558 with an aspartic acid and an arginine, respectively (n = 4-5/group). Controls were water-injected oocytes (n = 4). ** p < 0.01 compared to E-K.

Discussion
In this study, we examined the effects of Asp/Glu 555 and other charged residues in the entrance anion binding site S2 on Cl − selectivity and made the following observations. (i) Replacing Asp 555 in NBCe1 with a charge-conserved Glu induces a permissiveness to Cl − that is normally not a substrate. This replacement does not alter HCO3 − selectivity as INBC is favorably produced when both HCO3 − and Cl − are present. (ii) Under the Na + -free condition, D555E produces ICl even if HCO3 − is available in the bath. The reason is that the anion binding site is not occupied with HCO3 − in this condition; as a result, Cl − is accessible to the site. Thus, Na + is required for HCO3 − to access its binding site. (iii) The ICl induced by D555E is due to a charge interaction between Glu 555 and Lys 558 . Other Lys residues in site S2 have negligible effects on Cl − transport. Glu 555 and Lys 558 are not simultaneously present in any member of the SLC4A bicarbonate transporters, indicating that the high HCO3 − selectivity in these transporters is maintained by avoiding a charge interaction between the two residues. This molecular feature is interesting as it is generally understood that electrostatic interactions contribute to protein structure and create a suitable environment for protein function such as enzyme catalysis, protein-ligand binding, thermal stability, and macromolecular assemblies [21,24,25]. In this sense, our study provides novel evidence that the anion selection in the bicarbonate transporters is established by avoiding a specific interaction between residues in the anion binding site, rather than maintaining such interaction. The length in angstrom was determined using the molecular visualization program ChimeraX with the rotamer probability of higher than 0.05. A hydrogen bond was identified when the bond length was <4 Å. (C) Comparison of I Cl produced by mutations of Glu 555 and Lys 558 . E-K is the Glu 555 -Lys 558 pair (n = 7). N-K and Q-K are the replacement of Glu 555 with an asparagine and a glutamine, respectively (n = 5/group). E-E and E-R are the replacement of Lys 558 with an aspartic acid and an arginine, respectively (n = 4-5/group). Controls were water-injected oocytes (n = 4). ** p < 0.01 compared to E-K.

Discussion
In this study, we examined the effects of Asp/Glu 555 and other charged residues in the entrance anion binding site S2 on Cl − selectivity and made the following observations. (i) Replacing Asp 555 in NBCe1 with a charge-conserved Glu induces a permissiveness to Cl − that is normally not a substrate. This replacement does not alter HCO 3 − selectivity as I NBC is favorably produced when both HCO 3 − and Cl − are present. (ii) Under the Na + -free condition, D555E produces I Cl even if HCO 3 − is available in the bath. The reason is that the anion binding site is not occupied with HCO 3 − in this condition; as a result, Cl − is accessible to the site. Thus, Na + is required for HCO 3 − to access its binding site. (iii) The I Cl induced by D555E is due to a charge interaction between Glu 555 and Lys 558 . Other Lys residues in site S2 have negligible effects on Cl − transport. Glu 555 and Lys 558 are not simultaneously present in any member of the SLC4A bicarbonate transporters, indicating that the high HCO 3 − selectivity in these transporters is maintained by avoiding a charge interaction between the two residues. This molecular feature is interesting as it is generally understood that electrostatic interactions contribute to protein structure and create a suitable environment for protein function such as enzyme catalysis, protein-ligand binding, thermal stability, and macromolecular assemblies [21,24,25]. In this sense, our study provides novel evidence that the anion selection in the bicarbonate transporters is established by avoiding a specific interaction between residues in the anion binding site, rather than maintaining such interaction.
The amino acid residues in the chimeric transporter we examined correspond to Asp 555 , Lys 558 , and Lys 562 in NBCe1, all of which constitute site S2 located near the entrance of the ion accessibility pathway. Site S2 also contains Lys 559 , a DIDS-interacting residue [12], but we did not examine this residue for Cl − selectivity because it is conserved in all SLC4A transporters. The SLC4A transporters contain either Asp 555 or Glu 555 but maintain a high selectivity to HCO 3 − ; thus, a residue capable of interacting with these residues should not be conserved. The CryoEM of NBCe1 [11] shows that Asp 555 and Lys 558 are located to the protein center, while Lys 559 and Lys 562 are positioned further away from the center. The bond length between Asp 555 and Lys 558 is higher than the maximum length required for a salt bridge to take place, but Glu 555 substitution has decreased the length ( Figure 7A,B). We have previously demonstrated that D555E produces a large conductance in response to NO 3 − , which is structurally in a trigonal planar arrangement. The effective radius of NO 3 − is bigger than the molecular radius of Cl − (1.89 Å for NO 3 − vs. 1.81 Å for Cl − ), but D555E produces a larger NO 3 − current than I Cl . It is, thus, likely that site S2 is molded to sterically distinguish HCO 3 − or CO 3 2− from other polyatomic anions in a trigonal planar arrangement. The charge interaction between Glu 555 and Lys 558 in D555E modifies this steric arrangement in a way that other structurally similar ionic compounds, including NO 3 − , are allowed. The modified steric arrangement also allows Cl − to access the site but, given its monatomic molecule and competition with HCO 3 − or CO 3 2− , we think that a Cl − leak occurs at one of the three coordinating residues for peripheral oxygen atoms of HCO 3 − or CO 3 2− . This interpretation is consistent with the MD simulations that Lys 558 and Lys 559 are the closest coordinating residues of CO 3 2− , determined from ion density maps and contact frequency analysis.
The results from our study provide new insights into the mechanism underlying ion transport in NBCe1, in addition to anion selectivity. The Glu 555 -Lys 558 pair produces I Cl in the absence of CO 2 /HCO 3 − and I NBC in the presence of CO 2 /HCO 3 − ; that is, the presence or absence of I Cl reflects whether the anion binding site is occupied with HCO 3 − or CO 3 2− . I Cl is produced in Na + -free CO 2 /HCO 3 − ; thus, the binding site is not occupied in the absence of Na + , indicative of Na + precondition prior to anion binding. Based on this interpretation, a model of the ion binding process can be made. In NBCe1, ion transport begins with a recruitment of Na + to its binding site. The Na + binding then allows HCO 3 − or CO 3 2− to access its anion binding site and as a result both ions are bound to the transporter. The steric arrangement of Asp 555 , Lys 558 , and Lys 559 in site S2 is critical for distinguishing HCO 3 − or CO 3 2− from other anions. The same ion recruitment process also takes place in a mutant transporter containing the Glu 555 -Lys 558 pair, such as D555E. However, the charge interaction between the two residues modifies the steric arrangement of residues in S2, such that other anions, such as Cl − , are permissive; as a result, Cl − is accessible to the anion binding site. Our model proposes that Na + binding is a necessary first step prior to anion binding and, thus, should be independent of external HCO 3 − or CO 3 2− levels. In this sense, it is interesting to note that the apparent affinity of NBCe1 for Na + is independent of external HCO 3 − concentrations [26]. We think that the negatively charged Asp/Glu 555 facilitates Na + recruitment from the extracellular fluid surrounding the transporter. One might argue that Na + should overcome an electrostatic repulsion from the positively charged Lys residues before reaching its binding site. Decreased I NBC by E5558K and E558K/D562K ( Figure 5F) could be accounted for by the electrostatic repulsions from Lys residues. On the other hand, Yamazaki et al. [27] have reported that K558R, a single nucleotide polymorphism in human NBCe1, has a significantly reduced transport activity but no change in apparent Na + affinity. Either way, it is premature to conclude that Lys residues influence Na + recruitment to the binding site. The MD simulation model proposes that substrate ions transiently bind to site S2 and then move to site S1, which ultimately leads to a protein conformational change for ion translocation. The TM5-replaced chimeric transporter in this study contains NBCn1-S2 but still produces I NBC , indicative of electrogenic cotransport. Thus, I NBC can be induced, regardless of whether site S2 is molded for HCO 3 − transport in NBCn1 or HCO 3 − or CO 3 2− transport in NBCe1. Then, a question arises whether the charges in site S2 are critical for HCO 3 − or CO 3 2− recruitment. The chimeric transporter contains NBCe1-S1, indicating that the production of I NBC is determined by the anion that occupies site S1. We think that, whereas site S2 allows a transient binding of HCO 3 − or CO 3 2− , S1 determines which of the two anions is translocated via the transporter. Our interpretation further suggests that NBCn1-S2 can recruit CO 3 2− , in addition to HCO 3 − , although HCO 3 − is more favorably recruited. Nevertheless, it is difficult to envision how CO 3 2− is selected by both NBCn1-S2, which contains negatively charged residues, and NBCe1-S2, which contains positively charged residues. Additional studies are demanded to elucidate the role of site S2 in anion recruitment.
Does I Cl induced by the Glu 555 -Lys 558 pair represent a channel activity or transporter activity? If I Cl is a channel activity, we should then observe a current in response to HCO 3 − (I HCO3 ), comparable to I Cl in response to Cl − . However, we did not observe I HCO3 under the Na + -free condition. The lack of I HCO3 under the Na + -free condition reflects that HCO 3 − movement via D555E is solely mediated by electrogenic Na/HCO 3 transport that generates I NBC . The important finding is that I Cl is significantly inhibited by electrogenic Na/HCO 3 transport ( Figure 1B,C), indicating that I Cl competes with I NBC . Thus, I Cl is associated with a transporter activity. As described above, we envision that D555E modified the HCO 3 − binding site to produce an anion leak. On the other hand, I Cl can be produced without Na + , implicating a separate channel activity. This leads us to a conclusion that I Cl is associated with both transporter activity and channel activity, and they overlap. It is difficult to envision how the two activities overlap, and additional studies are required to address the exact nature of I Cl .
Lastly, our study leads us to a discussion about a pathological implication of Cl − leak mediated by mutations in NBCe1. Cl − and HCO 3 − movements tightly coordinated in many cells, and specific transporters and channels are involved in regulating such coordination [28][29][30][31]. Obviously, Cl − leak is undesirable in cells and tissues where NBCe1 is highly expressed and regulates HCO 3 − transport for cellular and physiological function. Myers et al. [32,33] have reported that Q913R, a mutation identified from a patient with proximal renal tubular acidosis, causes intracellular retention of NBCe1 and a gain of function activity in Cl − leak. It is expected that this mutation causes a depolarization in the basolateral membrane of renal proximal tubules; as a result, the driving force for HCO 3 − reabsorption is decreased. A Cl − leak via the mutation is also expected to alter the coupling of Cl − and HCO 3 − movement observed in secretory epithelia, such as pancreas and salivary glands [34]. Another mutation of interest is K558R that has a reduced transport activity [27]. Our analysis of the bond length between Asp 555 and Arg 558 in K558R is less than 4 Å (3.82 Å with the probability of 0.1 and 3.5 Å with the probability of 0.05), implicating a salt bridge between the two residues. It will be interesting to examine whether this mutation can cause Cl − leak. Additionally, depending upon NBCe1 variants, intracellular Cl − can regulate the transporter activity [35]. Thus, the lack of Cl − leak in NBCe1 is beneficial for cellular HCO 3 − homeostasis and epithelial electrolyte secretion. In summary, by analyzing the TM5 chimeric transporter and relevant point mutants, we identified a charge interaction in site S2 as a key factor for anion selectivity and provided new insights into CO 3 2− or HCO 3 − recruitment to the binding site and ion binding sequence. Future studies will be of the molecular mechanism underlying ion selectivity and translocation in other Na/HCO 3 transporters. and base line current were stabilized, solutions were switched to 5% CO 2 , 25 mM HCO 3 − (pH 7.4). The rate of pH change (dpH/dt) was determined by drawing a line during the first 4 min of recovery from CO 2 -induced acidification.

Salt Bridge Experiment
For assessment of salt bridges, an oocyte expressing the mutant transporters was clamped at 0 mV and superfused with 96 mM Na/gluconate (plus 5 mM mannitol) or 197 mM mannitol, plus a small amount of chloride (<3 mM), until base line currents became stable. Then, a series of test solutions containing 1, 10, 20, 40, and 96 mM of NaCl were applied. NaCl in each test solution replaced the equivalent amount of mannitol or Na/gluconate. Each test solution was bracketed with NaCl-free solution to maintain steady-state baseline between test solutions. The ionic strength (I) was determined using the equation: where Ci is the molar concentration of ion i (mol/L), and Zi is the charge number of that ion.

Analysis of Charge Interaction in Site S2
Analysis of the binding site S2 was performed with CryoEM structure of the human NBCe1 (accession code: 6CAA) from the RCSB Protein Data Bank using the molecular visualization program UCSF ChimeraX 1.1 [37]. A hydrogen bond between the side chain carboxy group of Asp 555 and amino group of nearby Lys residues was identified when the distance between them was <4 Å. For D555E or the TM5-replaced chimeric transporter, amino acid changes were analyzed using the structure editing function with Dunbrack rotamer library [22] built in ChimeraX. A hydrogen bond was identified from the rotamer probability of higher than 0.05.

Statistical Analysis
Data were reported as mean ± standard error. The level of significance was determined using (i) unpaired, two-tailed Student t-test for comparison between NBCe1 and D555E or chimeric protein; (ii) paired, one-tailed test for comparison of single transporters in two different solutions; (iii) one-way ANOVA for comparison of I Cl or I NBC among multiple mutants; and (iv) two-way ANOVA for comparison between I Cl vs. I NBC among multiple mutants. The p value of less than 0.05 was considered significant. Data were analyzed using Prism 7 (GraphPad; La Jolla, CA, USA) and Microsoft Office Excel add-in Analysis ToolPak (Redmond, WA, USA).